This invention relates to arrays, particularly biopolymer arrays such as DNA or protein arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
Polynucleotide arrays (such as DNA or RNA arrays) and peptide array, are known and may be used, for example, as diagnostic or screening tools. Such arrays include regions (sometimes referenced as spots or features) of usually different sequence polynucleotides or peptides arranged in a predetermined configuration on a substrate. The array is xe2x80x9caddressablexe2x80x9d in that different features have different predetermined locations (xe2x80x9caddressesxe2x80x9d) on a substrate carrying the array.
Biopolymer arrays can be fabricated using in situ synthesis methods or deposition of the previously obtained biopolymers. The in situ fabrication methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, and in U.S. Pat. No. 6,180,351 and WO 98/41531 and the references cited therein for polynucleotides. In situ methods also include photolithographic techniques such as described, for example, in WO 91/07087, WO 92/10587, WO 92/10588, and U.S. Pat. No. 5,143,854. The deposition methods basically involve depositing biopolymers at predetermined locations on a substrate which are suitably activated such that the biopolymers can link thereto. Biopolymers of different sequence may be deposited at different feature locations on the substrate to yield the completed array. Procedures known in the art for deposition of polynucleotides, particularly DNA such as whole oligomers or cDNA, are described, for example, in U.S. Pat. No. 5,807,522 (touching drop dispensers to a substrate), and in PCT publications WO 95/25116 and WO 98/41531, and elsewhere (use of an ink jet type head to fire drops onto the substrate).
In array fabrication, the quantities of DNA available for the array are usually very small and expensive. Sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require the manufacture and use of arrays with large numbers of very small, closely spaced features.
The arrays, when exposed to a sample, will exhibit a binding pattern. The array can be read by observing this binding pattern by, for example, labeling all targets such as polynucleotide targets (for example, DNA), in the sample with a suitable label (such as a fluorescent compound), scanning an illuminating beam across the array and accurately observing the fluorescent signal from the different features of the array. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample. Peptide or arrays of other chemical moieties can be used in a similar manner. Techniques and apparatus for scanning chemical arrays are described, for example, in U.S. Pat. Nos. 5,763,870 and 5,945,679. Apparatus which reads an array by scanning an illuminating beam by the foregoing technique are often referred to as scanners and the technique itself often referred to as scanning.
Array scanners typically use a laser beam as a light source, which is scanned over the array features. A detector (typically a fluorescence detector) with a very high light sensitivity is normally desirable to achieve maximum signal-to-noise in detecting hybridized molecules, particularly in array scanners used for DNA sequencing or gene expression studies,. At present, photomultiplier tubes (xe2x80x9cPMTsxe2x80x9d) are still the detector of choice although charge coupled devices (xe2x80x9cCCDsxe2x80x9d) can also be used. PMTs are typically used for temporally sequential scanning of array features, while CCDs permit scanning many features in parallel (for example, one line of features simultaneously, in which case an illuminating line may be used).
During scanning of an array, triplet saturation occurs. That is, fluorescent species are normally excited to a state from which they return to the singlet ground state while emitting the fluorescent light. However, the excited species has a finite probability of ending up in a lowest triplet state. Species in the triplet state do not emit fluorescence and thus are lost to producing a fluorescent signal while remaining in that state. Unfortunately, such triplet states may have very long lifetimes. Saturation is discussed in more detail, for example, in U.S. Pat. No. 5,945,679. As any region containing a fluorescent species may undergo multiple excitations during scanning, an increasing proportion will be unavailable to produce a signal due to saturation. A known solution to this problem is to re-scan a line on the array after waiting for a sufficient time for the fluorescent species to recover from saturation. However, the present invention realizes that for an array containing thousands of features, this may lengthen the scanning process. Furthermore, the present invention realizes that other problems may arise depending upon the scanning pattern. For example, in a rectangular scanning pattern a line is scanned from a first end to a second, and the next line is scanned from the second end to the first, and the process repeated for subsequent lines in turn. In this pattern, the time that a location has to recover from saturation is shorter toward the beginning and end of a scan line than at the center. The present invention realizes that this may lead to incomplete recovery from saturation and thus to a non-uniformity in detected signals from a uniform array.
It would be desirable then, to provide a means to scan an array in which the effect of saturation can be at least reduced.
The present invention then, provides a method of reading fluorescence signals from an array of chemical moieties (such as different sequence peptides or polynucleotides, for example different DNA sequences). In one such method multiple locations on the array are illuminated and any resulting fluorescence from the array is detected. In this case a later illuminated location is spatially closer to an earlier illuminated location than is a temporally intervening illuminated location lying on a same line as the later and earlier illuminated locations. This procedure may be repeated in one or more further cycles as required and using other locations, until the array or a desired portion of it has been read. Alternatively, multiple paths across the array may be illuminated and any resulting fluorescence from the array is detected. In such case the paths extend in a same lengthwise direction and are spaced from one another in a crosswise direction, and the spatial sequence of the paths does not correspond to their temporal sequence. For example, at least one later illuminated path may be closer to an earlier illuminated path than a temporally intervening illuminated path. The multiple paths may be parallel lines.
In the method, one or more later illuminated locations or paths may be interleaved between one or more previously illuminated locations or paths. The time between illuminating a location or path and illuminating a closest later illuminated location or path, may be selected based on a saturation characteristic of a fluorophore producing the fluorescence. Alternatively, the time may be based on an identifier associated with the array (such as being carried on an array substrate or a housing for the array), or on a spatial distribution of the illumination and a pixel size during the detecting.
Spacings of illuminated locations can be selected between any of the locations as desired. For example, the spacing between the spatially nearest locations or paths of the earlier, temporally intervening, and interleaved locations or paths may be equal. Further, various illumination techniques can be used within the methods of the present invention. For example, when the paths are lines they may be illuminated by scanning a light beam along them. As another example, timewise successively illuminated lines during any one cycle may be illuminated by scanning a light beam in the same or in opposite directions. The present invention further provides an apparatus which can execute a method of the present invention. The apparatus includes an illumination source to cause fluorescence of the chemical moieties. A scan system directs the illumination source to different locations on the array, while a detector detects any resulting fluorescence from the array. A processor controls the scan system to obtain the illumination of a method of the present invention. A computer program product for use with an apparatus, for reading fluorescence signals from an array of chemical moieties, is also provided to execute the steps of a method of the present invention.
While the methods and apparatus have been described in connection with arrays of various moieties, such as polynucleotides or DNA, other moieties can include any chemical moieties such as biopolymers.
The present invention can provide any one or more of the following or other benefits. For example, an array can be scanned such that loss of signal from saturation effects is kept low, or maintained at similar levels during the array scanning process.